We are interested in defining the structural components of RNA polymerase II and proteins that interact with this multimeric enzyme, and in characterizing their functional relationships. In yeast 12 subunits of the core enzyme have been identified and some fifty or more proteins have been demonstrated to interact with RNA polymerase II. We are using the fruit fly Drosophila melanogaster in a series of a classical and molecular genetic experiments to define the role of RNA polymerase II in regulating gene expression. Four subunits are cloned in Drosophila and three have been mutated. There is a high degree of conservation of RNA polymerase from bacteria to man. However, some of the answers we have obtained by studying RNA polymerase in a multicellular organism indicate that there is an increased complexity of the enzyme in higher eukaryotes. Many of the mutations we have identified in RNA polymerase II and interacting genes cause discrete mutant phenotypes suggesting that they are defective in only a subset of functions or steps required for transcription by RNA polymerase II. These mutations serve as the basis for our research project. This year we have finished an analysis of the effects of the elongation inhibitor alpha-amanitin on the homeotic transformation called the Ubx effect caused by certain alleles of RNA polymerase II. This work suggests that the transcriptional defect occurs prior to the formation of the open complex during initiation as alpha-amanitin does not alter the Ubx effect. We have also made significant progress on the cloning of the suppressor gene S3. We have begun to sequence a 6.6kb region thought to contain the transcribed portion of the gene though this clone cannot contain all the regulatory sequences as it does not provide normal S3 function when transformed back into flies. Two larger approximately 20kb fragments are now being injected to confirm the identity of this gene.